Despite decades of progress in cardiovascular biology, heart disease remains the leading cause of death in the developed world. Recently, therapies based on cardiac progenitor cells (CPCs) have emerged as promising potential cardiac therapeutics. However, we do not know the mechanisms underlying CRC self renewal, proliferation and differentiation, a prerequisite for cardiac regenerative therapy. My recent findings suggest that canonical Wnt signaling is required for CRC expansion. To search for downstream mediators of Wnt/p-catenin signaling, I performed expression array analyses of CRCs from mouse embryos with and without constitutively active Wnt/p-catenin signaling. This search revealed several genes with pivotal roles in CRC development that are negatively affected by p-catenin, including Isietl, Myocd and Smyd1. Notably, |3- catenin stabilization severely downregulated isietl, a temporal marker of undifferentiated CRCs that is required for normal cardiogenesis. /s/ef t-deficiency increased the number of CRCs and suppressed their cardiac differentiation. To understand how cellular decisions are implemented by Isietl and Wnt/p-catenin signaling, I have begun to identify the critical downstream pathways that affect CRC maintenance and differentiation. I found that Isietl loss-of-function severely compromised expression of Myocd, a gene required for smooth muscle differentiation. Interestingly, Isietl did not affect expression of Smyd1, an essential gene for cardiomyocyte differentiation, suggesting an Isietl-independent regulation of Stnydl by Wnt/p-catenin signaling. Among the genes upregulated by p-catenin, I found that Bhlhb2, a transcriptional repressor with a novel role in cardiogenesis, is expressed specifically in the cardiac region. Importantly, increased Bhihb2 \e\/e\s in CPCs resulted in downregulation of Smyd1, raising the possibility that Wnt/pcatenin signaling may regulate Smyd1 expression through Bhlhb2. Taken together, these findings set the stage for a mechanistic exploration of the role of Wnt/p-catenin signaling and Isietl in understanding maintenance and differentiation of CRCs that will facilitate future cell-based heart therapeutics. The specific aims of this proposal are (1) To test whether Isietl affects the self-renewal, proliferation, and differentiation of CRCs;(2) To determine if Isietl is an essential effector for Wnt/p-catenin signaling-mediated expansion of CRCs;(3) To investigate if Isietl and Wnt/p-catenin signaling directly activate Myoccf and Bhlhb2, respectively, to affect CRC fates. These studies will lay the foundation for understanding the molecular mechanisms of CRC maintenance and differentiation.